Title: The use of M-FISH to analysis the presence of genome instability in cells exposed to orthopaedic implant wear debris
1The use of M-FISH to analysis the presence of
genome instability in cells exposed to
orthopaedic implant wear debris
- Presented by Martin Figgitt
- Bristol Implant Research Centre
- Southmead Hospital
- Bristol
2Contents
- Introduction orthopaedic background, implant
types etc
- In vitro studies-metal ions
3Introduction
- Hip and knee orthopaedic implants the second
most common elective operation in UK
- More orthopaedic implants in younger eg 40 years
old
- Implants produce wear debris in both particulate
and metal ion form ( eg cobalt and chromium)
- Does any of this wear debris have any detrimental
effects upon patients eg genotoxicity
4The Bristol Study
- A comparison of the genotoxicity of two hip
resurfacing implants Birmingham and ASR
- The study is comprised of three components
1)Patients M-FISH performed on pre-operative and
post-operative blood samples (6 months, 1 year
and 2 year)
2)In-vitro study of nanoparticle wear debris
3)In-vitro study of metal ions associated with
wear debris
5ASR
Birmingham
Hip Resurfacing Orthopaedic Implants
6Outline of procedure
X-ray-Implant in situ
Hip Resurfacing- Principle
7The metal ions in vitro model
- Use primary human fibroblasts (BJ)
- Expose cells to metal ions (Chromium, Cobalt)
physiological concentrations
- Culture and harvest cells over a 30 day period
- M-FISH analysis (whole genome to be visualised)-
to investigate genome instability
8Experimental Outline
- Fibroblasts (BJ cell line) seeded at 2.5x 105 per
75 cm2 culture flask
- Cells left overnight to adhere, before exposure
metal ions
- Pulse exposure to cobalt, chromium III and
chromium VI ions
- One set of cultures exposed to the ions separately
- Second set with chromium III and chromium VI ions
in combination with cobalt ions
- M-FISH analysis of fibroblast metaphases
9METAL ION EXPOSURE 24 HOURS
CoCl2
1.3,25,50 ppb
Cr III
HARVESTING AND PASSAGING
2,20,40 ppb
DAY 1,5,10,15---------30
Cr VI
2,20,40 ppb
Cr IIICo
1.3/2,25/20,50/40 ppb
Cr VICo
1.3/2,25/20,50/40 ppb
M-FISH ANALYSIS
Control
Experimental Schematic
10True Colour
Labelling Scheme
Pseudo Colours
M-FISH Metaphase
11Translocation
Deletion
Fragment
Aneuploidy
M-FISH Chromosomal Aberrations
12Chromosome aberration levels
13Do these aberration levels change overtime?
What is the composition of the aberrations, is
there a pattern?
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18Summary
- Initial exposure to the metal ions induce
chromosomal aberrations
- Over the time period analysis metal ion induced
chromosomal aberrations regresses
- Both CrIII and Cr VI induce simple and complex
aneuploidy
- The combination of Cobalt with CrIII and CrVI
results in higher levels of chromosomal
aberrations
- Cr VI displays a higher incidence and persistence
of complex aneuploidy overtime with and without
the presence of cobalt ions
19Future Work
- Compare metal ion results with nanoparticle
experiments
- Investigate possible mechanisms causing
aneuploidy eg tubulin disruption
- Prolonged exposure experiments
- Investigations with cell lines defective in DNA
repair
20Acknowledgements
Dr CP Case Mr A Blom Professor I Learmonth Dr R
Newson
BIRC
Image Associates M-FISH technology
Grant Sponsors ARC
21Thank your for attention
Any questions?